PROJECT SUMMARY Fibroblast growth factor-23 (FGF23) is a hormone that induces urinary phosphate loss, vitamin D deficiency, and left ventricular hypertrophy. In chronic kidney disease (CKD) patients, FGF23 concentrations are markedly elevated, and associated with risk of heart failure (HF), mortality, and CVD events, making FGF23 a candidate factor linking CKD and cardiovascular disease (CVD). We are completing a phase 2 trial targeting FGF23 lowering, and are poised to implement a phase 3 trial based on the results. Yet, major unanswered questions remain about FGF23 metabolism, which directly influence clinical trial design. The strength of association of two FGF23 assays (the C-terminal and intact assays) with clinical outcomes differ markedly. Sample size estimates for a phase 3 trial vary from ~3,000 to ~23,000 individuals depending upon estimates driven by one assay or the other. This grant aims to understand the impact of key factors regulating FGF23 metabolism in humans, and in so doing, define the appropriate effect estimates to support power calculations to determine the feasibility and design of the pivotal phase 3 trial. The much stronger associations of the C-terminal FGF23 assay with CVD events raise major questions about the key regulators of FGF23 metabolism, and the role of unmeasured confounders in prior studies. The most likely sources of confounding in the prior literature are iron deficiency and inflammation, both morbidity risk factors themselves, and both induce higher levels of the C-terminal fragments but do not meaningfully impact intact FGF23 concentrations. Kidney function is another key determinant that may influence the relative concentrations of intact and C-terminal FGF23. Both forms of FGF23 are elevated in CKD, but not equivalently, as intact FGF23 increases disproportionately with declining eGFR. We hypothesize that the kidney is a critical organ for FGF23 clearance, and will clear C-terminal FGF23 fragments more efficiently than intact FGF23. In Aim 1, we will evaluate and compare the relative strengths of association of C-terminal and intact FGF23 with HF, mortality, and CVD events in an efficiently designed study within the Cardiovascular Health Study. We will also determine the degree to which iron deficiency and inflammation influence the relative strengths of association of C-terminal and intact FGF23 with these endpoints. In Aim 2, using specimens from 165 individuals who provided paired renal artery and vein specimens at the time of renal angiography, we will determine renal clearance of C-terminal and intact FGF23 and other markers of mineral metabolism. Collectively, we will determine the influence of iron deficiency, inflammation, and kidney function on FGF23 metabolism. In so doing, we will determine the strength of the independent associations of FGF23 with clinical endpoints, which will directly inform clinical trial design.